EP0805209A2 - Construction d'acides nucléiques contenant des gènes codant pour des signaux de transport - Google Patents

Construction d'acides nucléiques contenant des gènes codant pour des signaux de transport Download PDF

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EP0805209A2
EP0805209A2 EP97106531A EP97106531A EP0805209A2 EP 0805209 A2 EP0805209 A2 EP 0805209A2 EP 97106531 A EP97106531 A EP 97106531A EP 97106531 A EP97106531 A EP 97106531A EP 0805209 A2 EP0805209 A2 EP 0805209A2
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nucleic acid
promoter
acid construct
cell
activator
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EP0805209A3 (fr
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Hans-Harald Prof. Dr. Sedlacek
Rolf Prof. Dr. Müller
Reinhard Prof. Dr. Lührmann
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Sanofi Aventis Deutschland GmbH
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Hoechst AG
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the present application relates to nucleic acid constructs which can be used in genetic engineering and in particular in the prophylaxis or therapy of diseases (hereinafter referred to as gene therapy).
  • genes are introduced into the organism that are to be expressed in the organism.
  • the regulation of the expression of these genes is important for the prophylactic or therapeutic effect of gene therapy.
  • Patent applications PCT / GB95 / 02000, PCT / EP 95/03370, PCT / EP 95/03371, PCT / EP 95/03368, PCT / EP 95/03339 describe regulators for the expression of a gene. These regulators consist of an activator sequence, the function of which is, for example, the cell-specific or virus-specific activation of the basal transcription. The DNA sequence of this activator sequence is linked with its 3 'end to the 5' end of a promoter module. The structural gene is in turn linked to the 3 'end of the promoter module with its 5' end.
  • the promoter module consists of nucleic acid sequences for binding the transcription factors of the families CDF and CHF or E2F and CHF. This binding leads to an inhibition of the upstream activator sequence in the G0 and G1 phase of the cell cycle and thus to an inhibition of the transcription of the structural gene located downstream (i.e. in the direction of transcription).
  • the DNA content of the cell is in a diploid state.
  • the cell is at rest in the G0 phase, and its cell cycle progression is inhibited in the G1 phase.
  • the S phase follows the G1 phase, in where DNA synthesis takes place and where the genome is replicated.
  • This is followed by the G2 phase, in which the cell is in the tetraploid state.
  • the daughter cells come into the G0 or G1 state.
  • a cell-specific or virus-specific activator sequence with a promoter module that inhibits this activator sequence in the G0 and G1 phase thus enables the cell-specific or virus-specific as well as cell cycle-specific (i.e. limited to the S and G2 phase) regulation of the expression of a structural gene.
  • chimeric promoter The combination of an activator sequence with a promoter module is called a chimeric promoter.
  • chimeric promoter There are numerous uses for chimeric promoters in gene therapy, but there are also a number of limitations due to deficiencies.
  • nucleic acid constructs which enable precise regulation of the expression of foreign genes (transgenes) in the host cells.
  • the present invention therefore relates to nucleic acid constructs which have a nuclear retention signal which is linked downstream to a transgene in the reading direction.
  • the transcription product of the nuclear retention signal preferably has a binding structure for a nuclear export factor.
  • the first (I) promoter or enhancer sequence (a) and the second (II) promoter or enhancer sequence (d) can be the same or different and, at least one of components a) and d) can be non-specific, cell-specific, virus-specific, metabolic, can be activated in particular by hypoxia or cell cycle specific.
  • At least one of the promoter or enhancer sequences (a) and (d) can be a chimeric promoter, in which the promoter module CDE-CHR or E2FBS-CHR interact with an upstream neighboring cell-specific, virus-specific or metabolically activatable sequence and thereby can influence, in particular inhibit, the expression of a gene located downstream.
  • nucleic acid constructs in which the promoter or enhancer sequence (a) and / or (d) and / or the activator-responsive promoter is a chimeric promoter and the activator subunit (g) is a gene for at least one Represents transcription factor that activates the chimeric promoter of the activator-responsive promoter (h).
  • activator-responsive promoter activated by two activator subunits (g, g ') (h) is the LexA operator in conjunction with the SV40 promoter.
  • the activator subunit (g) comprises the cDNA for the LexA DNA binding protein coding for amino acids 1-81 or 1-202, the 3 'end of which is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435).
  • the second activator subunit (g ') comprises the cDNA of the Gal80 binding domain of the Gal4 protein coding for amino acids 851-881, the 3' end of which is linked to the 5 'end of the cDNA of the SV40 large T antigen coding for the Amino acids 126-132, the 3 'end of which is linked to the 5' end of the cDNA for the transactivation domain of the VP16 of HSV-1 coding for amino acids 406-488.
  • an activator-responsive promoter activated by two activator subunits is the binding sequence for the Gal4 protein in connection with the SV40 promoter.
  • the activation unit (g) comprises the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), the 3 'end of which is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435).
  • the second activation subunit (g ') comprises the cDNA for the Gal80 binding domain of Gal4 (amino acids 851 to 881), the 3' end of which is linked to the 5 'end of the cDNA of the nuclear localization signal of SV40 (SV40 Large T, amino acids 126 to 132), whose 3 'end is linked to the 5' end of the cDNA for the transactivation domain of VP16 of HSV-1 coding for amino acids 406-488.
  • the gene coding for the nuclear retention signal is preferably selected from the group comprising the Rev-responsive element (RRE) of HIV-1 or HIV-2, the RRE-equivalent retention signal of retroviruses or the RRE-equivalent retention signal of the HBV.
  • RRE Rev-responsive element
  • the nuclear export factor (s) is preferably a gene selected from the group comprising the Rev gene of the viruses HIV-1, HIV-2, Visna-Maedi virus, Caprine arthritis encephalitis virus, the virus of the infectious anemia of the horse Immunodeficiency virus of the cat, of retroviruses, of HTLV or the gene of the hnRNP-A1 protein or the gene of the transcription factor TFIII-A.
  • the nucleic acid is DNA.
  • the nucleic acid constructs according to the invention are usually used as vectors, in particular plasmid vectors or viral vectors.
  • the transgene is usually structural genes which code for a pharmacologically active substance which is selected from the group comprising cytokines, growth factors, antibodies or antibody fragments, fusion proteins between ligands such as antibodies or antibody fragments and cytokines or growth factors, receptors for Cytokines or growth factors, antiproliferative or cytostatic proteins, angiogenesis inhibitors, coagulation factors, thrombosis-inducing substances and anticoagulants, fibrinolytic substances, complement activating proteins, viral envelope proteins, bacterial antigens and parasitic antigens, proteins and ribozymes acting on the bloodstream.
  • the transgene is preferably a structural gene which codes for a ribozyme which inactivates the mRNA which codes for a protein selected from the group comprising cell cycle control proteins, in particular cyclin A, cyclin B, cyclin D1, cyclin E, E2F1-5 , cdc2, cdc25C or DP1, or virus proteins or cytokines or growth factors or their receptors.
  • the transgene can be a structural gene which codes for a protein which triggers controlled cell death (apoptosis), in particular sphingomyelinase.
  • the transgene (b) can be a structural gene which codes for an enzyme which cleaves a precursor of a drug into a drug.
  • the structural gene can code for a ligand-enzyme fusion protein, wherein the enzyme cleaves a precursor of a pharmaceutical into a pharmaceutical and the ligand binds to a cell surface, preferably to endothelial cells or tumor cells.
  • the promoter, enhancer or activator sequence can be selected from the group of gene regulatory nucleotide sequences activated in endothelial cells, smooth muscle cells, striated muscle cells, macrophages, lymphocytes, tumor cells, liver cells, leukemia cells and glial cells or from promoter sequences of the viruses HBV, HCV, HSV, HPV , EBV, HTLV or HIV.
  • the invention also relates to isolated cells or cell lines which contain a nucleic acid construct according to the invention.
  • Such cells can be used to provide a remedy for treating a disease, the provision of the remedy comprising introducing the nucleic acid construct into a target cell.
  • Such diseases are often accompanied by excessive cell proliferation.
  • nucleic acid construct must first be introduced into the target cell.
  • nucleic acid constructs according to the invention make it possible to use any promoters, enhancers or activator sequences and, in particular, to reinforce the fate of the transcription product of the structural gene introduced into the cell or to increase the intracellular transport of the transcription product of the structural gene introduced into the cell.
  • FIG. 1 The arrangement of the individual components is shown in FIG. 1, for example.
  • the nucleic acid construct according to the invention for the enhanced intracellular transport of the transcription product has, in addition to a promoter or enhancer sequence I which activates the basal transcription of the structural gene, and a transgene which codes for the desired active substance, a nuclear retention signal (NRS), the cDNA of which at the 5'-end is linked to the 3'-end of the structural gene and its messenger RNA is binding structure for a nuclear export factor (NEF).
  • NNS nuclear retention signal
  • NEF nuclear export factor
  • nucleic acid constructs have a further promoter or enhancer sequence (II) which activates the basal transcription of the cDNA of the nuclear export factor (NEF) and binds its expression product to the nuclear retention signal (NRS) and thereby the transport of the transcription product Structural genes mediated from the cell nucleus.
  • II further promoter or enhancer sequence
  • FIG. 2 The arrangement of the individual components is shown in FIG. 2, for example.
  • the promoter or enhancer sequences of components a) or d) can be the same or different, furthermore components d) and e) can be located upstream or downstream of components a), b) and c).
  • At least one strong promoter or enhancer sequence such as from CMV (EP-A-0173177) or SV40 or any other promoter or enhancer sequence known to the person skilled in the art, is preferably used as the promoter or enhancer sequence.
  • At least one promoter or enhancer sequence can be activated cell-specifically, metabolically (e.g. by hypoxia), virus-specific or cell-cycle-specific in the nucleic acid constructs according to the invention.
  • At least one promoter or enhancer sequence is a chimeric promoter in the nucleic acid constructs according to the invention.
  • a chimeric promoter is the combination of an upstream cell-specific, metabolically or virus-specific activator sequence with a downstream promoter module.
  • the promoter module is characterized by a nucleotide sequence which binds the transcription factors of the CDF and CHF or E2F and CHF families and thereby can inhibit activation of the upstream activator sequence in the G0 and G1 phases of the cell cycle (Lucibello et al., EMBO J. 14, 132 (1994), PCT / GB95 / 02000).
  • At least one promoter or enhancer sequence (component a) or d)) is an activator-responsive promoter unit in the nucleic acid constructs according to the invention.
  • FIG. 3 The arrangement of the individual components of a preferred activator-responsive promoter unit is shown in FIG. 3.
  • FIG. 4 The insertion of a preferred activator-responsive promoter unit into a nucleic acid construct according to the invention is shown, for example, by FIG. 4.
  • activator-responsive promoter units can represent, for example, chimeric promoter constructs according to the scheme in FIG. 5.
  • activator-responsive promoter units according to the invention can represent binding sequences for chimeric transcription factors from DNA binding domains, protein-protein interaction domains and transactivation domains.
  • Preferred structural genes for a pharmacologically active substance are genes for ribozymes, ribozymes with combined antisense RNA, proteins and glycoproteins, selected from the group comprising cytokines, growth factors, receptors for cytokines or growth factors, fusion proteins from ligands (eg antibodies or antibody fragments) and cytokines or growth factors antiproliferative or cytostatic proteins, angiogenesis inhibitors, thrombosis-inducing proteins, anticoagulants, complement-activating proteins, enveloping substances of viruses and enveloping substances of bacteria.
  • Genes for ribozymes which specifically cleave the mRNA of genes which code for proteins which are particularly involved in controlling the cell cycle are particularly preferred.
  • the nuclear retention signal is a nucleotide sequence which hinders the transport of a pre messengerger RNA linked to it through the nuclear membrane, but which, on the other hand, represents a binding structure for an export protein.
  • This export protein mediates the transport of the NRS containing premessenger or messenger RNA from the cell nucleus into the cytoplasm. A pre messengerger or messenger RNA containing the NRS is thus removed from the cell nucleus by binding to the export protein (Fischer et al., Cell 82, 475 (1995)).
  • the NRS is preferably the Rev Responsive Element (RRE) sequence of retroviruses.
  • RRE Rev Responsive Element
  • this RRE is a sequence comprising 243 nucleotides (nucleotides 7362-7595; Muesing et al., Nature 313, 450 (1985)) in the env gene (Malim et al., Nature 338, 254 (1989); Kjems et al., PNAS 88, 683 (1991)).
  • nuclear retention signal in the sense of the invention can also be a homologous and / or functionally similar (analog) nucleotide sequence, for example the RRE-equivalent element of the HBV virus (Huang et al., Mol Cell Biol. 13 , 7476 (1993)).
  • the nuclear export factor is (NEF) a nucleotide sequence which codes for a protein which binds to the mRNA of the NRS and which mediates the transport of the premessenger RNA or messenger RNA containing an NRS from the cell nucleus into the cytoplasm (or from the cytoplasm into the cell nucleus).
  • NEF nuclear export factor
  • the rev gene of retroviruses especially of the HIV-1 or HIV-2 virus (Daly et al., Nature 342, 816 (1989); Emerman et al., Cell 57, 1155 (1989) ; Felber et al., PNAS 86, 1495 (1989); Fischer et al., EMBO J. 13, 4105 (1994)).
  • the rev protein of the rev gene of retroviruses binds with its N-terminal domain (Zapp et al., Nature 342, 714 (1989); Malim et al., Cell 65, 241 (1991)) to the RRE in the pre mRNA (Iwai et al., Nucl. Acids Res. 20, 6465 (1992)).
  • the binding between the RRE and the rev protein enables the transport of "nonspliced" premessenger RNA, but also any other RNA which contains an RRE, from the cell nucleus into the cytoplasm (Fischer et al., EMBO J. 13, 4105 (1994); Fischer et al., Cell 82, 475 (1995)) and thus significantly increased translation.
  • nucleotide sequences which encode proteins which are homologously and functionally similar to the rev protein of HIV-1 can be used as NEF (Bogerd et al., Cell 82, 485 (1995)), such as, for example, the rev Gene of the Visna-Maedi virus (VMV; Tiley et al., J. Virol. 65, 3877 (1991)) or the rev gene of the Caprine arthritis encephalitis virus (CAEV; Tiley et al., J. Virol. 65, 3877 (1991)).
  • VMV Visna-Maedi virus
  • CAEV Caprine arthritis encephalitis virus
  • genes which code for proteins which have little or no homology to the rev protein but are functionally similar to the rev protein of HIV-1.
  • HTLV-1 HTLV-1
  • EIAV equine infectious anemia virus
  • FV cat immunodeficiency virus
  • the NEF can also be Act nucleotide sequences for proteins that cause RNA to be removed from the nucleus without being retained in the nucleus by an NRS.
  • proteins include, for example, the transcription factor TFIIIA (Gaddat et al., Cell 60, 619 (1990)) or the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1 protein; Pinol-Roma et al., Nature 355, 730 (1992)).
  • nuclear transport proteins in the broader sense include the "heat shock protein 70" (HSC70; Mandell et al., J. Cell Biol. 111, 1775 (1990)) or the protein kinase inhibitor CPKI (Fantozzi et al., J Biol. Chem. 269, 2676 (1994), Wen et al., J. Biol. Chem. 269, 32214 (1994)).
  • HSC70 heat shock protein 70
  • CPKI protein kinase inhibitor
  • nucleic acid constructs preferably consist of DNA.
  • nucleic acid constructs is understood to mean artificial structures made of nucleic acid which can be transcribed in the target cells. They are preferably inserted into a vector, plasmid vectors or viral vectors being particularly preferred.
  • the nucleic acid constructs according to the invention can be used to express a transgene (component b) both cell-specifically or virus-specifically or under certain metabolic conditions and also cell-cycle-specifically, the structural gene preferably being a gene which codes for a pharmacologically active substance or else for an enzyme which splits an inactive precursor of a drug into an active drug.
  • the structural gene can be chosen so that this enzyme as a fusion protein with a Ligand is expressed and this ligand binds to the surface of cells, for example proliferating endothelial cells or tumor cells.
  • the present invention also relates to cells of yeasts or mammals which contain a nucleic acid construct according to the invention.
  • the nucleic acid constructs are introduced into cell lines, which can then be used after transfection to express the transgene. These cells can thus be used to provide a remedy for patients as well as in the patient and thus for the treatment of a disease.
  • a preferred use of the nucleic acid construct according to the invention is the treatment of a disease, the provision of the remedy comprising the introduction of a nucleic acid construct into a target cell and its virus- or target cell-specific and cell cycle-specific expression.
  • the disease is often one that is accompanied by excessive cell proliferation, the preparation of the remedy comprising the introduction of a nucleic acid construct into a target cell and its virus- or target cell-specific expression at the stage of cell proliferation.
  • nucleic acid constructs according to the invention do not occur in nature in this form, i.e. the transgene or structural gene for the drug or for an enzyme or for a ligand-enzyme fusion protein is not naturally combined with the nuclear retention signal (NRS) and neither are naturally connected to promoter I and this combination is again not naturally combined with the nucleotide sequence consisting of promoter II and the nuclear export factor (NEF).
  • NSS nuclear retention signal
  • NEF nuclear export factor
  • the promoters I and II and the structural gene for the active ingredient (or for the enzyme) of the nucleic acid constructs according to the invention are selected depending on the intended use.
  • nucleic acid constructs Depending on the planned use of the nucleic acid constructs, the following embodiments can be selected:
  • the preferred promoters include or Activator sequences from promoters or enhancers are those gene-regulatory sequences or elements for genes which code for proteins which are particularly detectable in endothelial cells (or in cells in the immediate vicinity of proliferating endothelial cells).
  • genes which have mutations for the ⁇ inactivation sites of the expressed proteins without the function being impaired thereby.
  • the retinoblastoma protein (pRb) and the related p107 and p130 proteins are inactivated by phosphorylation.
  • a pRb / p110 -, p107 - or p130 cDNA sequence is thus preferably used which is point-mutated in such a way that the phosphorylation sites of the encoded protein are replaced by non-phosphorylatable amino acids.
  • DNA sequences of fusion proteins between the cytokines or growth factors listed on the one hand and ligands for receptors on the cell membrane can also be used as active substance in the sense of the invention Find.
  • ligands for receptors on the cell membrane such as, for example, an antibody specific for endothelial cells or tumor cells or the Fc part of the human immunoglobulin
  • DNA sequence for an enzyme is also to be understood as an anti-tumor or anti-inflammatory substance, which is capable of converting precursors of an anti-tumor active substance into an anti-tumor active substance.
  • the homologous signal sequence contained in the DNA sequence can be replaced by a heterologous signal sequence which improves the extracellular discharge.
  • the signal sequence of the ⁇ -glucuronidase (DNA position ⁇ 27 to 93; Oshima et al., PNAS 84, 685 (1987)) can be replaced by the signal sequence for the immunoglobulin (DNA position ⁇ 63 to ⁇ 107; Riechmann et al ., Nature 332, 323 (1988)) or by the signal sequence for the CEA (DNA position 33 33 to 134 134; Schrewe et al., Mol. Cell. Biol. 10, 2738 (1990), Berling et al., Cancer Res.
  • DNAs of such enzymes are preferred, which are stored to a lesser extent in lysosomes by point mutation and increasingly secreted.
  • point mutations have been described, for example, for ⁇ -glucuronidase (Shiplex et al., J.Biol.Chem. 268, 12193 (1993).
  • a sequence for a transmembrane domain can be introduced as an alternative or in addition to the signal sequence.
  • the transmembrane sequence of the human macrophage colony-stimulating factor (DNA position ⁇ 1485 to ⁇ 1554; Cosman et al., Behring Inst. Mitt. 83, 15 (1988)) or the DNA sequence for the signal and transmembrane region of the human respirator syncytial virus (RSV) glycoprotein G (amino acids 1 to 63 or their partial sequences, amino acids 38 to 63; Vijaya et al., Mol. Cell Biol. 8, 1709 (1988), Lichtenstein et al., J. General Virol .
  • RSV respirator syncytial virus
  • the DNA sequence for the signal and transmembrane region of the influenza virus neuraminidase (amino acids 7 to 35 or the partial sequence amino acids 7 to 27; Brown et al., J. Virol. 62, 3824 (1988 )) between the DNA sequence for the promoter and the DNA sequence for the enzyme (eg the ⁇ -glucuronidase).
  • the nucleotide sequence can be at the 3 'end of the promoter and immediately before the 5' end of the start signal (ATG) of the signal or transmembrane sequence GCCACC or GCCGCC (Kozak, J. Cell. Biol. 108, 299 (1989).
  • nucleotide sequence for a glycophospholipid anchor can also be used to anchor the enzyme in the cell membrane of the cells forming the enzyme be inserted.
  • a glycophospholipid anchor is inserted at the 3 'end of the nucleotide sequence for the enzyme and can also be used to insert a signal sequence.
  • Glycophospholipid anchors are, for example, for the CEA (DNA position ⁇ 893 to ⁇ 1079; Berling et al., Cancer Res. 50, 6534 (1990)), for the N-CAM (Cunningham et al., Science 236, 799 ( 1987) and for other membrane proteins, such as, for example, Thy-1 (Clissold, Biochem. J. 281, 129 (1992)) or CD16 (Selvaray et al., Nature 333, 565 (1988)).
  • CEA DNA position ⁇ 893 to ⁇ 1079; Berling et al., Cancer Res. 50, 6534 (1990)
  • N-CAM Cunningham et al., Science 236, 799 ( 1987)
  • other membrane proteins such as, for example, Thy-1 (Clissold, Biochem. J. 281, 129 (1992)) or CD16 (Selvaray et al., Nature 333, 565 (1988)).
  • Another way of anchoring enzymes to the cell membrane according to the present invention is to use a DNA sequence for a ligand-enzyme fusion protein.
  • the specificity of the ligand of this fusion protein is directed against a membrane structure located on the cell membrane of proliferating endothelial cells or of tumor cells.
  • the ligands that bind to the surface of proliferating endothelial cells include, for example, antibodies or antibody fragments directed against membrane structures of endothelial cells, as described, for example, by Burrows et al. (Pharmac. Ther. 64, 155 (1994)), Hughes et al. (Cancer Res. 49, 6214 (1989)) and Maruyama et al. (PNAS-USA 87, 5744 (1990)).
  • these include antibodies against the VEGF receptors.
  • the murine monoclonal antibodies are preferably used in humanized form. Humanization takes place in the method described by Winter et al. (Nature 349, 293 (1991)) and Hoogenbooms et al. (Rev.Tr.Transfus.Hemobiol. 36, 19 (1993)). Antibody fragments are produced according to the state of the art, for example in the Winter et al. (Nature 349, 293 (1991)), Hoogenboom et al. (Ref.Tr.Transfus.Hemobiol. 36, 19 (1993); Girol.Mol.Immunol. 28, 1379 (1991) or Huston et al. (Intern.Rev.Immunol. 10, 195 (1993))).
  • the ligands also include all active substances which bind to membrane structures or membrane receptors on endothelial cells.
  • these include substances that contain mannose, IL-1 or growth factors or their fragments or partial sequences from you that bind to receptors expressed by endothelial cells, such as PDGF, bFGF, VEGF, TGF ⁇ (Pusztain et al., J Pat. 169: 191 (1993)).
  • This also includes adhesion molecules that bind to activated and / or proliferating endothelial cells. Adhesion molecules such as SLex, LFA-1, MAC-1, LECAM-1 or VLA-4 have already been described (reviews by Augustin-Voss et al., J. Cell Biol. 119, 483 (1992), Pauli et al., Cancer Metast. Rev. 9, 175 (1990), Honn et al., Cancer Metast. Rev. 11, 353 (1992)).
  • the ligands also include antibodies or their fragments which are directed against tumor-specific or tumor-associated antigens on the tumor cell membrane.
  • DNA sequence for a ribozyme which is able to cleave and thereby inactivate the transcription product (messenger RNA) of a gene for a cell cycle control protein is also to be understood as an anti-tumor or anti-inflammatory substance.
  • Preferred substrates for such ribozymes is the messenger RNA of the genes of cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, cdc25C and DP1.
  • the invention further relates to nucleic acid constructs in which a combination of the DNA sequences of several of the same antitumor or anti-inflammatory substances (A, A) or different anti-tumor substances (A, B) is present.
  • A, A antitumor or anti-inflammatory substances
  • A, B anti-tumor substances
  • IRS internal ribosome entry site
  • IRES IRES have been described, for example, by Mountford and Smith (TIG 11, 179 (1995), Kaufman et al., Nucl. Acids Res. 19, 4485 (1991), Morgan et al., Nucl. Acids Res. 20, 1293 (1992) , Dirks et al., Gene 128, 247 (1993), Pelletier and Sonenberg, Nature 334, 320 (1988) and Sugitomo et al., BioTechn. 12, 694 (1994).
  • the cDNA of the IRES sequence of the poliovirus (position ⁇ 140 to
  • such an active ingredient has additive (A + A, A + B1) or synergistic effect in the sense of the invention.
  • An active substance in the sense of the invention is a DNA sequence, the expressed protein of which causes the proliferation and / or differentiation of blood cells.
  • the active substance in the sense of the invention is the DNA sequence for a cytokine, a chemokine, a growth factor or one of its inhibitors, for a ribozyme catalytically for the transcription product of one of these DNA sequences or for the transcription product of a gene which is responsible for a cell cycle control protein or a DNA Sequence for an antibody or an antibody fragment or for an enzyme or for a fusion protein of an antibody, cytokine or growth factor with an enzyme.
  • the selection of the active substance depends on the underlying disease to be treated and the chosen promoter sequence.
  • promoters preference is given to using promoters, activator sequences from promoters or enhancers or gene-regulatory sequences of those genes with which transcription factors in active endothelial cells, Synovial cells and inflammatory cells interact.
  • the preferred promoter sequences include gene regulatory sequences or elements from genes which code for proteins which are particularly expressed in endothelial cells, synovial cells and inflammatory cells.
  • An active substance in the sense of the invention is a DNA sequence, the expressed protein of which directly or indirectly inhibits inflammation, for example in the joint, and / or promotes the reconstitution of extracellular matrix (cartilage, connective tissue) in the joint.
  • Vaccines are used to prevent infectious diseases.
  • the possibilities of producing effective vaccines by conventional means are limited (Brown, Int. J. Technol. Assessm. Health Care 10, 161 (1994)), Ellis, Adv. Exp. Med. Biol. 327, 263 (1992) ), Arnon et al., FASEB J. 6, 3265 (1992)).
  • Active substances for the prophylaxis of infectious diseases in the sense of this invention are distinguished by a high degree of safety because of their cell specificity and cell cycle regulation.
  • Promoter sequences of cell genes are selected as the activator sequence, the activity of which is changed particularly by infections with bacteria or parasites, or promoter sequences of those viruses are to be selected which transform the cells infected by them and stimulate proliferation.
  • viruses include, for example, HBV, HCV, HSV, HPV, HIV, EBV and HTLV.
  • the active substance to be selected is the DNA of a protein which has cytostatic, cytotoxic or antiviral effects. Examples of cytotoxic or cytostatic proteins have already been listed above. When an enzyme is selected, the precursor of an antiviral cytotoxic or antiparasitic substance which can be cleaved by this enzyme is subsequently administered.
  • DNA sequences for fusion proteins between the cytokines, growth factors or the extracellular part of the receptors on the one hand and a ligand on the other can also be used as active substance in the sense of the invention, for example fusion proteins with the Fc part of the human immunoglobulin in the EPA 0 464 633 A1.
  • Ribozymes which digest the mRNA of genes for cell cycle control proteins or the mRNA of viruses also count as active substances. Ribozymes catalytically for HIV have been clearly described, for example, by Christoffersen et al., J. Med. Chem. 38, 2033 (1995).
  • an active substance in the sense of this invention is the DNA sequence for an antibody of a specificity which inactivates the respective virus or its fragments containing VH and VL or its VH and VL fragments connected via a linker, produced, for example, in accordance with the method of Marasco et al . (Proc. Natl. Acad. Sci. ⁇ USA 90, 7889 (1993)). Examples of antibodies of such specificity against viruses are given in Section 8.4. listed.
  • Such active substances within the meaning of the invention also include the DNA of an anti-idiotype antibody or its antigen-binding fragments, whose antigen binding structures, the "complementarity determining regions", represent copies of the protein or carbohydrate structure of the neutralizing antigen of the infectious agent.
  • anti-idiotype antibodies can replace carbohydrate antigens in particular in bacterial infectious agents.
  • the invention further relates to an active ingredient in which a combination of the DNA sequences of the same active substances (A, A) or different active substances (A, B) is present.
  • the cDNA of an "internal ribosome entry site" (IRES) is preferably interposed as a regulatory element.
  • IRES IRES have been described, for example, by Montford and Smith (TIG 11, 179 (1995), Kaufman et al., Nucl. Acids Res. 19, 4485 (1991), Morgan et al., Nucl. Acids Res. 20, 1293 (1992) , Dirks et al., Gene 128, 247 (1993), Pelletier and Sonnenberg, Nature 334, 320 (1988) and Sugitomo et al., BioTechn. 12, 694 (1994).
  • the cDNA of the IRES sequence of the poliovirus (positions ⁇ 140 to
  • such an active ingredient has additive (A + A, A + B1) or synergistic effect in the sense of the invention.
  • two identical or two different antiviral active substances can be combined with one another for the therapy of viral diseases.
  • active substances which code for different antigens of an infectious agent or different infectious agents can be combined with one another.
  • the active substance which codes for the antigen of an infectious agent can be combined with an active substance which codes for a cytokine or a cytokine receptor.
  • the cytokines or cytokine receptors which thus form (after injection of the active ingredient) simultaneously with the infectious agent antigen can influence the type and strength of the developing immune reaction.
  • DNA sequences for cytokines and cytokine receptors which enhance the humoral immune response have already been described under 6.2.d), those for enhancing the cellular immune response under 6.2.a) and 6.2.c).
  • a gene regulatory nucleotide sequence is provided as the promoter or activator sequence, with which transcription factors, formed or actively interacting in leukemia cells or tumor cells, interact.
  • the preferred promoters or activator sequences include gene regulatory sequences or elements from genes which code for proteins formed especially in leukemia cells or tumor cells.
  • An active substance in the sense of the invention is to be understood as a DNA sequence, the expressed protein of which directly or indirectly inhibits the proliferation of cells, in particular also of leukemia cells or tumor cells.
  • active substances include, for example, the DNA sequences for inhibitory, cytostatic, apoptosis-inducing or cytotoxic proteins or of enzymes, as have already been described.
  • the active substance is also to be understood as the DNA sequence for a ribozyme which catalyzes the cleavage of the mRNA of the genes for cell cycle control proteins.
  • promoters or activator sequences from promoters or enhancers in the sense of the invention preference is given to using gene regulatory sequences or elements from genes which, particularly in smooth muscle cells, code for protein formed.
  • An active substance in the sense of the invention is to be understood as a DNA sequence whose expressed protein inhibits the proliferation of smooth muscle cells.
  • These proliferation inhibitors include the proteins already mentioned under 2.2.a) and 2.2.c).
  • the active substance is also the DNA sequence for an enzyme that converts an inactive precursor of a cytostatic into a cytostatic (see 2.2.e)).
  • DNA sequence for a ribozyme specific for the mRNA of genes for cell cycle control proteins is also to be understood as an active substance (see 2.2.f)).
  • Preferred promoters or activator sequences for the purposes of the invention to use gene regulatory sequences or elements from genes which encode detectable proteins in smooth muscle cells, in activated endothelial cells, in activated macrophages or in activated lymphocytes.
  • proteins which are formed particularly in activated endothelial cells are described by Burrows et al. (Pharmac. Ther. 64, 155 (1994)).
  • these proteins, which occur more frequently in endothelial cells include, for example, proteins such as those already mentioned and the promoter sequences of their genes.
  • An activator sequence in the sense of this invention is also understood to mean promoter sequences of the genes for proteins which are formed to an increased extent in the immune reaction in macrophages and / or in lymphocytes. Such proteins have already been listed.
  • a DNA sequence which encodes a protein which directly or indirectly inhibits platelet aggregation or a blood coagulation factor or stimulates fibrinolysis is to be used as the active substance for inhibiting coagulation in the sense of this invention.
  • Anticoagulants are genes for, for example, plasminogen activators (PA), such as tissue PA (tPA) or urokinase-like PA (uPA) or protein C, antithrombin III, C-1S inhibitor, ⁇ 1-antitrypsin, the tissue factor pathway inhibitor (TFPI) or hirudin.
  • PA plasminogen activators
  • tPA tissue PA
  • uPA urokinase-like PA
  • TFPI tissue factor pathway inhibitor
  • hirudin hirudin.
  • a DNA sequence which encodes a protein which promotes blood coagulation directly or indirectly is to be used as the active substance for promoting coagulation in the sense of this invention.
  • proteins include, for example, factor VIII, factor IX or factor XIII of blood coagulation.
  • these include the promoter sequences for the genes of endothelial cell-specific proteins.
  • a preferred activator sequence is further to be understood as a nucleotide sequence (promoter or enhancer sequence) which is formed to a particular extent with transcription factors or which actively interacts in glial cells.
  • neurospecific factors are understood to be a DNA sequence which codes for a neuronal growth factor.
  • an activator-responsive promoter unit Production and testing of an activator-responsive promoter unit, the activator responsive promoter being the binding sequence for LexA.
  • the activator-responsive promoter unit according to the invention consists of the following different nucleotide sequences which follow one another downstream:
  • the sequence of the nucleotide sequences of the activator-responsive promoter units is shown in FIG. 13.
  • the promoter is now at its 3 'end with the sequence GCCACC (Kocak, J. Cell. Biol. 108, 229 (1989)) and this with the cDNA for the signal peptide of the immunoglobulin (nucleotide sequence ⁇ 63 to ⁇ 107; Riechmann et al., Nature 332, 323 (1988)). This is followed by the cDNA of ⁇ -glucuronidase (nucleotide sequence 93 93 to 1982 1982; Oshima et al., PNAS USA 84, 685 (1987)) according to FIG. 15.
  • the nucleotide construct thus produced is cloned into pUC18 / 19 or bluescript-derived plasmid vectors which are used directly or in colloidal dispersion systems for in vivo application.
  • the individual components of the construct are linked via suitable restriction sites, which are carried over to the termini of the various elements via PCR amplification.
  • the linkage takes place with the aid of enzymes and DNA ligases which are known to the person skilled in the art and are specific for the restriction sites. These enzymes are commercially available.
  • the described plasmid is used to transfect cultured human umbilical cord endothelial cells and fibroblasts (Wi-38) using a method known to the person skilled in the art (Lucibello et al., EMBO J. 14, 132 (1995)) and the amount of ⁇ -glucuronidase produced by the Endothelial cells measured using 4-methylumbelliferyl- ⁇ -glucuronide as a substrate.
  • endothelial cells are synchronized by withdrawing methionine over 48 hours in G0 / G1 (Nettelbeck et al., Publication in Preparation).
  • the DNA content of the cells is determined after staining with Hoechst 33258 in the fluorescence activation cell sorter (Lucibello et al., EMBO J. 14, 132 (1995)).
  • Transfected endothelial cells express significantly more ⁇ -glucuronidase than non-transfected endothelial cells.
  • the activator-responsive promoter unit described leads to cell-specific, cell cycle-dependent expression of the structural gene ⁇ -glucuronidase.
  • An active substance according to the present invention after local application, for example at the site of the tumor or after intracranial or subarachnoid administration or systemic, preferably intravenous or intraarterial administration, enables the activator-responsive promoter unit to be predominantly, if not exclusively, due to the cell cycle and endothelial cell specificity.
  • This ⁇ -glucuronidase cleaves an now well-tolerated doxorubicin- ⁇ -glucuronide (Jacquesy et al., EPO 0511 917 A1) into the cytostatic doxorubicin.
  • the active ingredient promises a high level of safety through both its cell and cell cycle specificity, it can also be used in high doses and, if necessary to be used several times at intervals of days or weeks for the therapy of tumor diseases.
  • the multiple promoter according to the invention consists of the following different nucleotide sequences which follow one another downstream:
  • nucleotide sequences of element A and element B are linked according to the scheme in FIG. 16.
  • the nucleotide construct thus produced is cloned into pUC18 / 19 or bluescript-derived plasmid vectors which are used directly or in colloidal dispersion systems for in vivo application.
  • the individual components of the construct are linked via suitable restriction sites, which are carried over to the termini of the various elements via PCR amplification.
  • the linkage takes place with the aid of enzymes and DNA ligases which are known to the person skilled in the art and are specific for the restriction sites. These enzymes are commercially available.
  • the described plasmid is used to transfect cultured human umbilical cord endothelial cells and fibroblasts (Wi-38) using a method known to the person skilled in the art (Lucibello et al., EMBO J. 14, 132 (1995)) and the amount of ⁇ -glucuronidase produced by the Endothelial cells measured using 4-methylumbelliferyl- ⁇ -glucuronide as a substrate.
  • endothelial cells are synchronized by withdrawing methionine over 48 hours in G0 / G1 (Nettelbeck et al., Publication in preparation).
  • the DNA content of the cells is determined after staining with Hoechst 33258 in the fluorescence activation cell sorter (Lucibello et al., EMBO J. 14, 132 (1995)).
  • Transfected endothelial cells express significantly more ⁇ -glucuronidase than non-transfected endothelial cells.
  • the multiple promoter unit described thus leads to cell-specific, cell cycle-dependent expression of the structural gene ⁇ -glucuronidase.
  • the activator-responsive promoter unit according to the invention consists of the following different nucleotide sequences which follow one another downstream:
  • the sequence of the nucleotide sequences of the activator-responsive promoter units is shown in FIG. 17.
  • the promoter is now at its 3 'end with the sequence GCCACC (Kocak, J. Cell Biol. 108, 229 (1989)) and this with the cDNA for the signal peptide of the immunoglobulin (nucleotide sequence ⁇ 63 to ⁇ 107; Riechmann et al ., Nature 332, 323 (1988)). This is followed by the cDNA of ⁇ -glucuronidase (nucleotide sequence 93 93 to 1982 1982; Oshima et al., PNAS USA 84, 685 (1987)) according to FIG. 19.
  • the nucleotide construct thus produced is cloned into pUC18 / 19 or bluescript-derived plasmid vectors which are used directly or in colloidal dispersion systems for in vivo application.
  • the individual components of the construct are linked via suitable restriction sites, which are carried over to the termini of the various elements via PCR amplification.
  • the linkage takes place with the aid of enzymes and DNA ligases which are known to the person skilled in the art and are specific for the restriction sites. These enzymes are commercially available.
  • the described plasmid is used to transfect cultured human umbilical cord endothelial cells and fibroblasts (Wi-38) using a method known to those skilled in the art (Lucibello et al., EMBO J. 14, 132 (1995)) and the amount of ⁇ -glucuronidase produced by the Endothelial cells measured using 4-methylumbelliferyl- ⁇ -glucuronide as a substrate.
  • endothelial cells are synchronized by withdrawing methionine over 48 hours in G0 / G1 (Nettelbeck et al., Publication in preparation).
  • the DNA content of the cells is determined after staining with Hoechst 33258 in the fluorescence activation cell sorter (Lucibello et al., EMBO J. 14 , 132 (1995)).
  • Transfected endothelial cells express significantly more ⁇ -glucuronidase than non-transfected endothelial cells.
  • the activator-responsive promoter unit described leads to cell-specific, cell cycle-dependent expression of the structural gene ⁇ -glucuronidase.
  • This activator-responsive promoter unit consists of the following different nucleotide sequences which follow one another downstream:
  • the nucleotide construct thus produced is cloned into the pXP2 plasmid vector (Nordeen, BioTechniques 6, 454 (1988)), which are used directly or in colloidal dispersion systems for in vivo application.
  • the individual components of the construct are linked via suitable restriction sites, which are carried over to the termini of the various elements via PCR amplification.
  • the linkage takes place with the aid of enzymes and DNA ligases which are known to the person skilled in the art and are specific for the restriction sites. These enzymes are commercially available.
  • the described plasmid is used to transfect cultured 3T3 fibroblasts using a method known to those skilled in the art (Lucibello et al., EMBO J. 14, 132 (1995)) and the amount of luciferase produced by the fibroblasts as described by Herber et al. (Oncogene 9, 1295 (1994)) and Lucibello et al. (EMBO J. 14, 132 (1995)).
  • the fibroblasts are synchronized by withdrawing serum in G0 / G1 for 48 hours.
  • the DNA content of the cells is determined after staining with Hoechst 33258 in the fluorescence activation cell sorter (Lucibello et al., EMBO J. 14, 132 (1995)).
  • a significant increase in luciferase can be determined in transfected fibroblasts compared to non-transfected fibroblasts.
  • the activator-responsive promoter unit described leads to cell cycle-dependent expression of the reporter gene luciferase.
  • An active substance according to the present invention after local application, for example at the site of the tumor or after intracranial or subarachnoid administration or systemic, preferably intravenous or intraarterial administration, enables the activator-responsive promoter unit to be predominantly, if not exclusively, due to the cell cycle and endothelial cell specificity. Only secrete proliferating endothelial cells ⁇ -glucuronidase. This ⁇ -glucuronidase cleaves an now well-tolerated doxorubicin- ⁇ -glucuronide (Jacquesy et al., EPO 0511 917 A1) into the cytostatic doxorubicin. This inhibits endothelial cell proliferation and has a cytostatic effect on these cells as well as on neighboring tumor cells. This leads to the inhibition of tumor growth.

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CA2204332A1 (fr) 1997-11-03
DE19617851A1 (de) 1997-11-13
AU716178B2 (en) 2000-02-17
EP0805209A3 (fr) 1998-04-22
ZA973802B (en) 1997-11-03
KR970074935A (ko) 1997-12-10
US6235526B1 (en) 2001-05-22
AU1998297A (en) 1997-11-06
CN1170040A (zh) 1998-01-14

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